In Project 3, we propose to exploit our understanding of cancer biology to develop assays for compounds that could lead to more effective and less toxic chemotherapeutic agents. We propose two general approaches to identifying new agents. First, we will develop assays that are not targeted to a specific cellular pathway, but instead search for general perturbations in cell growth, division, morphology, or embryonic development. By not specifying that agents act through a specific biological mechanism, this approach may provide the greatest change of identifying potential lead compounds that act through a novel mechanism, as many potential cellular targets are screened simultaneously. To complement this open-ended approach, we also propose to exploit our current understanding of cancer biology and basic cell biology to design assays that target specific proteins or biological mechanisms whose pharmacologic inhibition would be expected to be toxic to proliferating cells. Selected targets include gamma-tubulin, the cyclin B proteolysis system, and a mitotic kinesin (KCM) present in dividing cells, but not neurons. Inhibitors of the latter protein should lack the neurotoxicity seen in other tubulin inhibitors. We will also design assays to identify compounds that sensitize cancer cells to current chemotherapeutic agents and ionizing radiation. This will include a screen for inhibitors of the spindle assembly checkpoint, which may make cells more sensitive to anti- mitotic agents, and a screen for inhibitors of DNA glycosylases, which should make cells more sensitive to chemotherapies that damage DNA. These assays will make use of the small molecule libraries developed in Project 1. An important goal is to adapt these assays to the nanodroplet format being developed in Project 2, increasing the efficiency of the screening process. We will also utilize the target identification strategies being developed in Project 2 to identify the protein targets of small molecules for which the target has not been determined by the nature of the assay. Small molecules with potential activity will be resynthesized by the Synthetic Chemistry Core (Core B). The synthetic chemistry core will also support the generation of structural derivatives of the lead compound once its activity is confirmed following resynthesis. The Administrative Core A will coordinate the efforts of this Project with the work of Projects 1 and 2, and will provide administrative assistance and financial oversights.